Arthropod-borne viruses (arboviruses) face a two-fold challenge in countering the antiviral defenses of both vertebrate hosts and arthropod vectors. In vertebrates, the front-line defense against viruses is the interferon (IFN) response, whereas in many arthropods, it is RNA interference (RNAi), the targeted destruction of messenger-sense RNA with homology to a double-stranded RNA trigger. While the ability of arboviruses to induce and elude the IFN response has been an area of intense study, much less is known about the interactions between arboviruses and the arthropod RNAi response. It is particularly important to investigate this interaction in the mosquito-borne flaviviruses, which include a number of important emerging pathogens such as dengue, yellow fever and West Nile virus. Currently there is considerable excitement about the potential for harnessing RNAi using synthetic short interfering RNA's (siRNA's) to control flavivirus infection and transmission, but nothing is known about the ability of flaviviruses to inhibit RNAi, nor has the role of RNAi in control of natural vector infections been studied. Additionally, there are currently no antivirals available to treat any flavivirus, and proteins that act as viral suppressors of RNAi (VSR's) may offer novel targets for drug development. Vaccines are needed for many flaviviruses, particularly dengue virus, and mutations that disable VSR's may contribute to attenuation of vaccine candidates. Finally, a fundamental understanding of the pathways that vectors use to control flaviviruses, and the mechanisms arboviruses use to counter them, are needed to interpret patterns of flavivirus evolution and vector specificity. The proposed research will investigate three aspects of the interaction between dengue virus and RNAi in its mosquito vector by: (i) identifying which, if any, DENV proteins act as a viral suppressor of RNAi, (ii) measuring the impact of RNAi on DENV replication various mosquito systems, and (iii) monitoring the impact of RNAi on DENV genomic evolution. PUBLIC HEALTH RELEVANCE Dengue virus is a mosquito-transmitted pathogen that causes millions of cases of severe disease worldwide every year because there are no vaccines to prevent dengue infection and no antiviral medications to treat dengue disease. A newly-discovered pathway that blocks viral infection, called RNA interference, holds promise in the development of new antiviral therapies. The proposed research will investigate whether dengue virus possesses mechanisms to counteract RNA interference.